We propose to study DNA synthesis of parvovirus H-1 as a model for DNA synthesis in animal cells. The studies will include (1) purification of a soluble system from uninfected cells that converts H-1 viral single strands to replicative form (RF) DNA; (2) characterization of the protein covalently bound to the 5' termini of H-1 RF DNA; (3) analysis of the structures of replicative intermediates; and (4) development of a soluble system for in vitro replication of double-stranded RF DNA. Second, we propose to study the ability of the mechanism for synthesis of the RNA primer for animal cell DNA synthesis, to accept deoxynucleotides, nucleotide analogues and noncomplementary bases. The studies will be carried out both in vitro and in vivo and will include an assessment of possible effects of the misincorporation on DNA synthesis. Third, we propose to study the consequences of expansion of intracellular deoxyuridylate pools and contraction of thymidylate that result from treatment of cells with FdUrd. The studies will include (1) measurement of the intracellular pools for dUTP, FdUTP and dTTP; (2) measurement of the misincorporation of dUMP and FdUMP into DNA; and (3) analysis of the relationship between misincorporation/removal of Ura and FUra, DNA fragmentation, and cell death.